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SCIENTIFIC OPINION ADOPTED: 28 November 2019 doi: 10.2903/j.efsa.2019.5951 Evaluation of calcium lignosulfonate as a acceptable previous cargo for edible fats and oils EFSA Panel on Contaminants in the Food Chain (CONTAM), Dieter Schrenk, Margherita Bignami, Laurent Bodin, James Kevin Chipman, Jes us del Mazo, Bettina Grasl-Kraupp, Christer Hogstrand, Laurentius (Ron) Hoogenboom, Jean-Charles Leblanc, Carlo Stefano Nebbia, Elsa Nielsen, Evangelia Ntzani, Annette Petersen, Salomon Sand, Tanja Schwerdtle, Heather Wallace, Konrad Grob, Laurence Castle, Anna Christodoulidou and Christiane Vleminckx Abstract Shipping of edible fats and oils into Europe is permitted in bulk tanks, provided that the previous cargo is included in a positive list. The European Commission requested EFSA to evaluate the acceptability of calcium lignosulfonate as previous cargo for fats and oils. The evaluation was based on the same criteria as those used for the evaluation of the substances currently on the list in the Annex to Commission Directive 96/3/EC as a acceptable previous cargoes for edible fats and oils. In 2017, the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) concluded that calcium lignosulfonate did not meet the acceptability criteria, due to uncertainties as regards the composition and toxicity of its low-molecular weight fraction (LMWF) below 1,000 Da. In the current evaluation, new information, showing lack of genotoxicity of the LMWF isolated from a technical grade of calcium lignosulfonate was provided. Due to uncertainties regarding the presence of lignosulfonate components below 200 Da in this LMWF tested for genotoxicity, the CONTAM Panel concluded that the information provided was insufcient to assess the acceptability of calcium lignosulfonate as previous cargo. The Panel recommends a better analysis of the LMWF and a new genotoxicity test using this LMWF, including components < 200 Da, and evidence that the tested material is representative of the LMWF in products intended to be shipped as previous cargo for edible fat and oils. © 2019 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. Keywords: acceptable previous cargo, edible fats and oils, sea transport, calcium lignosulfonate Requestor: European Commission Question Number: EFSA-Q-2019-00130 Correspondence: [email protected] EFSA Journal 2019;17(12):5951 www.efsa.europa.eu/efsajournal

Transcript of Evaluation of calcium lignosulfonate as a acceptable ... · calcium lignosulfonate as previous...

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SCIENTIFIC OPINION

ADOPTED: 28 November 2019

doi: 10.2903/j.efsa.2019.5951

Evaluation of calcium lignosulfonate as a acceptableprevious cargo for edible fats and oils

EFSA Panel on Contaminants in the Food Chain (CONTAM),Dieter Schrenk, Margherita Bignami, Laurent Bodin, James Kevin Chipman, Jes�us del Mazo,

Bettina Grasl-Kraupp, Christer Hogstrand, Laurentius (Ron) Hoogenboom,Jean-Charles Leblanc, Carlo Stefano Nebbia, Elsa Nielsen, Evangelia Ntzani, Annette Petersen,

Salomon Sand, Tanja Schwerdtle, Heather Wallace, Konrad Grob, Laurence Castle,Anna Christodoulidou and Christiane Vleminckx

Abstract

Shipping of edible fats and oils into Europe is permitted in bulk tanks, provided that the previous cargo isincluded in a positive list. The European Commission requested EFSA to evaluate the acceptability ofcalcium lignosulfonate as previous cargo for fats and oils. The evaluation was based on the same criteriaas those used for the evaluation of the substances currently on the list in the Annex to CommissionDirective 96/3/EC as a acceptable previous cargoes for edible fats and oils. In 2017, the EFSA Panel onContaminants in the Food Chain (CONTAM Panel) concluded that calcium lignosulfonate did not meet theacceptability criteria, due to uncertainties as regards the composition and toxicity of its low-molecularweight fraction (LMWF) below 1,000 Da. In the current evaluation, new information, showing lack ofgenotoxicity of the LMWF isolated from a technical grade of calcium lignosulfonate was provided. Due touncertainties regarding the presence of lignosulfonate components below 200 Da in this LMWF tested forgenotoxicity, the CONTAM Panel concluded that the information provided was insufficient to assess theacceptability of calcium lignosulfonate as previous cargo. The Panel recommends a better analysis of theLMWF and a new genotoxicity test using this LMWF, including components < 200 Da, and evidence thatthe tested material is representative of the LMWF in products intended to be shipped as previous cargofor edible fat and oils.

© 2019 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalfof European Food Safety Authority.

Keywords: acceptable previous cargo, edible fats and oils, sea transport, calcium lignosulfonate

Requestor: European Commission

Question Number: EFSA-Q-2019-00130

Correspondence: [email protected]

EFSA Journal 2019;17(12):5951www.efsa.europa.eu/efsajournal

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Panel members: Dieter Schrenk, Margherita Bignami, Laurent Bodin, James Kevin Chipman, Jes�usdel Mazo, Bettina Grasl-Kraupp, Christer Hogstrand, Laurentius (Ron) Hoogenboom, Jean-CharlesLeblanc, Carlo Stefano Nebbia, Elsa Nielsen, Evangelia Ntzani, Annette Petersen, Salomon Sand, TanjaSchwerdtle, Heather Wallace and Christiane Vleminckx.

Acknowledgements: EFSA wishes to thank the following for the support provided to this scientificoutput: Marco Binaglia.

Suggested citation: EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), Schrenk D,Bignami M, Bodin L, Chipman JK, del Mazo J, Grasl-Kraupp B, Hogstrand C, Hoogenboom LR, Leblanc J-C, Nebbia CS, Nielsen E, Ntzani E, Petersen A, Sand S, Schwerdtle T, Wallace H, Grob K, Castle L,Christodoulidou A and Vleminckx C, 2019. Scientific Opinion on the evaluation of calcium lignosulfonateas a acceptable previous cargo for edible fats and oils. EFSA Journal 2019;17(12):5951, 17 pp. https://doi.org/10.2903/j.efsa.2019.5951

ISSN: 1831-4732

© 2019 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalfof European Food Safety Authority.

This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License,which permits use and distribution in any medium, provided the original work is properly cited and nomodifications or adaptations are made.

The EFSA Journal is a publication of the European FoodSafety Authority, an agency of the European Union.

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Summary

Shipping of edible fats and oils into Europe is permitted in bulk tanks, provided that the previouscargo is included in a positive list. The European Commission requested the European Food SafetyAuthority (EFSA) to evaluate the acceptability of calcium lignosulfonate as previous cargo for fats andoils. The evaluation was based on the criteria adopted by EFSA in 2009 and also used for theevaluation of the substances currently on the list of acceptable previous cargoes for edible fats and oilsin the Annex to Commission Directive 96/3/EC.

Calcium lignosulfonate was re-evaluated on the basis of new information provided after therecommendations made by the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) in2017 on the need for additional information, mainly regarding the composition and toxicity of the low-molecular weight fraction (LMWF) in the grades of lignosulfonate intended as previous cargo.

New data on the genotoxicity of a LMWF, isolated from a technical calcium lignosulfonate productas intended for shipping, were provided by the producer Borregaard. The tests comprised a reversemutation assay in bacteria and an in vitro micronucleus assay in human peripheral lymphocytes. Inconclusion, the LMWF was negative in the two genotoxicity tests.

Borregaard provided also data on the molecular mass distribution for: the LMWF used in thegenotoxicity testing; the raw calcium lignosulfonate product from which this LMWF was isolated; atypical product intended for the previous cargo; and the lignosulfonate 45–60 product purified byultrafiltration, that was evaluated in the past as food and feed additives. The molecular massdistribution of the LMWF deviated from that of the reference products; components < 200 Da werelargely absent in the LMWF.

In addition, Borregaard provided an Ames test and an acute toxicity tests on some commerciallignosulfonate products. In the absence of (a) further information of the product tested and ademonstration of its similarity with the products intended to be shipped and (b) adequate set ofin vitro genotoxicity assays, the relevance of these toxicity data for the evaluation of calciumlignosulfonate was questionable.

The Panel concluded that a better analysis of the, toxicologically most relevant, LMWF includingcomponents < 200 Da, is needed for calcium lignosulfonate to meet the criteria for acceptance asprevious cargo. In particular, mass calibration should be performed with lower molecular massstandards, and a more comprehensive detection, should be used. Genotoxicity tests in accordancewith EFSA guidance (Ames test and in vitro micronucleus test) (EFSA Scientific Committee, 2011, 2017,2019) should be performed using the LMWF, including components < 200 Da. The tested materialshould be representative of the LMWF in products intended to be shipped as previous cargo for ediblefat and oils.

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Table of contents

Abstract................................................................................................................................................... 1Summary................................................................................................................................................. 31. Introduction................................................................................................................................... 51.1. Background and Terms of Reference as provided by the requestor..................................................... 51.1.1. Background ................................................................................................................................... 51.1.2. Terms of Reference ........................................................................................................................ 51.2. Interpretation of the Terms of Reference.......................................................................................... 51.3. Additional information..................................................................................................................... 51.3.1. Criteria set for the for the evaluation of substances proposed as a acceptable previous cargoes........... 51.3.2. Legislation ..................................................................................................................................... 52. Data and methodologies ................................................................................................................. 62.1. Data.............................................................................................................................................. 62.1.1. Documents submitted to EFSA for the re-evaluation of calcium lignosulfonate..................................... 62.1.2. Data retrieved by search................................................................................................................. 72.2. Methodologies................................................................................................................................ 73. Assessment.................................................................................................................................... 83.1. Production and use of calcium lignosulfonate ................................................................................... 83.2. Previous evaluations ....................................................................................................................... 83.3. Current evaluation.......................................................................................................................... 93.3.1. Additional information received........................................................................................................ 93.3.2. Composition of the low molecular weight fraction used for toxicity studies.......................................... 93.3.3. Toxicity testing ............................................................................................................................... 114. Conclusion ..................................................................................................................................... 115. Recommendations .......................................................................................................................... 12Documentation as provided to EFSA (if appropriate) ................................................................................... 12References............................................................................................................................................... 12Abbreviations ........................................................................................................................................... 13Appendix A – In vitro genotoxicity with the LMWF ...................................................................................... 14Appendix B – Other toxicity tests with technical grades of lignosulfonate ...................................................... 15Annex A – Review of the criteria for acceptable previous cargoes for edible fats and oils (EFSA, 2009) ........... 16

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1. Introduction

1.1. Background and Terms of Reference as provided by the requestor

1.1.1. Background

Reg. (EC) 852/2004 on the hygiene of foodstuffs foresees that bulk food has to be transported incontainers/tanks reserved for food. However, Reg. (EU) No 579/2014 foresees for some exemptions. Itindicates that the bulk transport in seagoing vessels of liquid oils or fats, shall be permitted in tanksthat are not exclusively reserved for the transport of foodstuffs, provided that the previous cargoes arelisted in the Annex to the Regulation on the basis of an EFSA opinion.

In its opinion of 24 November 20161 on the evaluation of substances as a acceptable previouscargoes for edible fats and oils, EFSA evaluated 4 substances for their acceptability as previouscargoes for edible fats and oils. It concluded that calcium lignosulfonate does not meet theacceptability criteria as there are uncertainties regarding the composition and toxicity of the lowmolecular mass fraction. On 16 January 2019, the company Borregaard submitted to the Commissionand EFSA two new genetic toxicity studies on a low molecular weight fraction of calcium lignosulfonateand requested a re-evaluation of calcium lignosulfonate for acceptability as previous cargoes for fatsand oils. EFSA is now asked to evaluate this new information in order to verify whether the conclusionson calcium lignosulfonate in the previous opinion need to be updated.

1.1.2. Terms of Reference

In accordance with Art 29 (1) of Regulation (EC) No 178/2002, the European Commission asks theEuropean Food Safety Authority for an updated scientific opinion on the evaluation of substances as aacceptable previous cargoes for edible fats and oils taking into account the new information submittedby Borregaard or, in case the conclusions of the opinion would remain the same, to publish an opinionon the evaluation of the new data and the confirmation of the previous conclusions.

1.2. Interpretation of the Terms of Reference

No new information has been retrieved or provided that would change the previous conclusions ofthe CONTAM Panel (EFSA CONTAM Panel, 2017) in relation to other substances accepted as previouscargoes, except for calcium lignosulfonate. Therefore, the present opinion of the CONTAMPanel should comprise the re-evaluation of the substance calcium lignosulfonate (CAS No 8061-52-7)following the additional information and data provided to EFSA.

The evaluation is based on the criteria used for the Scientific Opinions on the evaluation of thesubstances currently on the list in the Annex to Commission Directive 96/3/EC as a acceptable previouscargoes for edible fats and oils.

1.3. Additional information

1.3.1. Criteria set for the evaluation of substances proposed as a acceptableprevious cargoes

In 2009, the CONTAM Panel review the criteria for acceptable previous cargoes for edible fats andoils set by the Scientific Committee on Food (SCF) (EFSA CONTAM Panel, 2009). The conclusions ofthe CONTAM Panel are summarised in Annex A.

1.3.2. Legislation

Council Directive 93/43/EEC2 on the hygiene of foodstuffs laid down the general rules of hygienefor foodstuffs and the procedures for verification of compliance with these rules. Chapter IV of theAnnex considered the hygienic transportation of foodstuffs. Commission Directive 96/3/EC3 derogatedcertain provisions of Council Directive 93/43/EEC on the hygiene of foodstuffs as regards the transport

1 EFSA Panel on Contaminants in the Food Chain (CONTAM); Scientific Opinion on the evaluation of substances as acceptableprevious cargoes for edible fats and oils. EFSA Journal 2017;15(1):4656.

2 Council Directive 93/43/EEC of 14 June 1993 on the hygiene of foodstuffs. OJ L 175, 19.7.1993, p. 1–11.3 Commission Directive 96/3/Euratom, ECSC, EC of 26 January 1996 granting a derogation from certain provisions of CouncilDirective 93/43/EEC on the hygiene of foodstuffs as regards the transport of bulk liquid oils and fats by sea. OJ L 21,27.1.1996, p. 42–46.

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of bulk oils and fats by sea, permitting the transport by sea of bulk oils and fats in tanks which havepreviously been used to transport substances listed in the Annex to this Directive and subject toconditions which ensure the protection of public health and safety of foodstuffs concerned.

In order to take account of scientific developments and based on the evaluations carried out by theSCF (1997, 2003), the list of acceptable previous cargoes was amended by Commission Directive 2004/4/EC4. Council Directive 93/43/EEC was repealed by Regulation (EC) No 852/20045 on the hygiene onfoodstuffs, which laid down general hygiene requirements relating to transport of food applicable to allfood business operators. Annex II of Chapter IV states, among others, that ‘receptacles in vehicles and/orcontainers are not to be used for transporting anything other than foodstuffs where this may result incontamination’.

The outcome of the EFSA scientific opinions on the evaluation of the substances on theiracceptability as previous cargoes for fats and oils published from 2009 to 2012 was taken into accountfor the subsequent repeal of Directive 96/3/EC and its replacement by Commission Regulation (EU) No579/20146. This Regulation provides for derogation from point 4 of Chapter IV of Annex II to Regulation(EC) No 852/2004 as regards the transport on seagoing vessels of oils and fats intended for or likely tobe used for human consumption under certain conditions and includes a revised list of acceptableprevious cargoes. As the combined entry for ‘ammonium nitrate solution and calcium nitrate (CN-9)solution and their double salt ‘created confusion to ship charterers and competent authorities, theCommission replaced this entry in the list of acceptable previous cargoes by the separate entries of‘ammonium nitrate solution’ and ‘calcium ammonium nitrate’. Further, having identical hazard profilesand only differing in the amount of crystal water, ‘calcium ammonium nitrate’, ‘calcium (II) nitratedehydrate’ and ‘calcium nitrate tetrahydrate’ were inserted. At its 68th plenary meeting,7 the EFSACONTAM Panel confirmed that these changes have no impact on the toxicological properties andchemical reactivity. Therefore, the list of acceptable previous cargoes in the Annex to Regulation (EU)No 579/2014 has been amended by Commission Regulation (EU) 2016/2388. On the basis of the EFSAscientific opinion of 4 November 2016,9 by means of Regulation (EU) No 2019/97810, 2-ethylhexanoland methylacetate were added to the Annex to Regulation (EU) No 579/2014. However, EFSA concludedthat calcium lignosulfonate does not meet the acceptability criteria, as there are uncertainties as regardsthe composition and toxicity of the low molecular mass fraction. Therefore, calcium lignosulfonate wasnot included in the Annex to Regulation (EU) No 579/2014 by means of Regulation (EU) 2019/978.

2. Data and methodologies

2.1. Data

2.1.1. Documents submitted to EFSA for the re-evaluation of calciumlignosulfonate

Borregaard AS, one of the main producers of lignosulfonate products, provided the CONTAMPanel with information on the lower molecular weight fraction (LMWF) < 1,000 Da of a calciumlignosulfonate product estimated to be representative of the products intended to be shipped asprevious cargo (see Documentation provided to EFSA No 1–3).

4 Commission Directive 2004/4/EC amending Directive 96/3/EC granting a derogation from certain provisions of Council Directive93/43/EEC on the hygiene of foodstuffs as regards the transport of bulk liquid oils and fats by sea. OJ L 15, 22.1.2004, p. 25–30.

5 Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of foodstuffs. OJL 139, 30.4.2004, p. 1–54.

6 Commission Regulation (EU) No 579/2014 of 28 May 2014 granting derogation from certain provisions of Annex II toRegulation (EC) No 852/2004 of the European Parliament and of the Council as regards the transport of liquid oils and fats bysea. OJ L 160, 29.5.2014, p. 14–20.

7 Minutes of the 68th CONTAM Plenary meeting available online: http://www.efsa.europa.eu/en/events/event/141125b-m.pdf10Commission

8 Regulation (EU) 2016/238 of 19 February 2016 amending the Annex to Regulation (EU) No 579/2014 granting derogationfrom certain provisions of Annex II to Regulation (EC) No 852/2004 of the European Parliament and of the Council as regardsthe transport of liquid oils and fats by sea. OJ L 45, 20.2.2016. p. 1–2.

9 EFSA Journal 2017;15(1):4656.10 Commission Regulation (EU) 2019/978 of 14 June 2019 amending the Annex to Regulation (EU) No 579/2014 granting

derogation from certain provisions of Annex II to Regulation (EC) No 852/2004 of the European Parliament and of the Councilas regards the transport of liquid oils and fats by sea. OJ L 159, 17.6.2019, p. 26–28.

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2.1.2. Data retrieved by search

The CONTAM Panel considered the previous assessments (EFSA CONTAM Panel, 2011, 2017) ascomprehensive, covering all relevant publications up to 18 April 2016. For the present evaluation of thesubstances as a acceptable previous cargoes, the CONTAM Panel considered literature made publiclyavailable after 18 April 2016. No new information was retrieved from PubMed, Web of Science or theEuropean Chemicals Agency (ECHA).

2.2. Methodologies

In the years 2009–2016, the CONTAM Panel evaluated the acceptability of the substances listed inthe Annex to Commission Directive 96/3/EC as a acceptable previous cargoes for edible fats and oils,based on its review of the criteria set by SCF (EFSA CONTAM Panel, 2009) and the experience gainedin its subsequent evaluations, which highlighted the importance of addressing impurities that might bepresent (EFSA CONTAM Panel, 2011, 2012a,b, 2017).

The present opinion is based on the criteria established by the EFSA CONTAM Panel (EFSA, 2009):

• The substance is transported/stored in an appropriately designed system, with adequatecleaning routines, including the verification of the efficacy of cleaning between cargoes,effective inspection and recording procedures. The CONTAM Panel was of the opinion thatrecords of the three previous cargoes should be kept, in accordance with the CodexRecommended International Code of Practice for the Storage and Transport of Edible Fats andOils in Bulk. The CONTAM Panel noted that the choices made with respect to design of thetransport system and the cleaning methods are part of the responsibility of those managingthe transport of previous cargoes. It was the nature and amount of substances that might becarried over into a subsequent cargo of edible fats and oils that was taken into account by theCONTAM Panel in its evaluation of previous cargoes.

• Residues of the substance in the subsequent cargo of fat or oil should not result in adversehuman health effects. The acceptable daily intake (ADI) (or total daily intake (TDI)) of thesubstance should be greater than or equal to 0.1 mg/kg body weight (bw) per day. Substancesfor which there is no numerical ADI (or TDI) should be evaluated on a case-by-case basis. Fornon-genotoxic substances, their transport as second and third previous cargoes is not ofconcern, taking into account their very limited carry over. However, genotoxic substanceswould not be acceptable as second and third previous cargoes.

• The substance should not be or contain a known allergen, unless the identified allergen can beadequately removed by subsequent processing of the fat or oil for its intended use. Thiscriterion covers all allergens, not only food allergens.

• If the substance reacts with edible fats and oils, reaction products must comply with the abovetwo criteria. Reactions may be promoted by the acidity from free fatty acids and may occurover many months or at high temperatures during refining after the transport; they do notneed to result in high yields to be potentially relevant.

• The development of analytical methods of sufficient sensitivity to check for residues in fatsand oils should be feasible, e.g. for control authorities, but such methods are seldomavailable, since most substances used as previous cargoes are not commonly analysed in fatsand oils. The CONTAM Panel therefore evaluated the feasibility of developing suchmethodology as part of its assessment of each substance. In those cases where, due to thenature or composition of the substance to be evaluated as previous cargo, the feasibility ofdeveloping suitable analytical methods was considered questionable, this was indicated whendiscussing the substance and was used as an argument for the rejection of a substance asprevious cargo.

• It is unrealistic to assume that chemical analysis would regularly be applied to check the purityof a substance used as previous cargo or the efficiency of the cleaning procedure. Therefore,the substances were evaluated under worst case assumptions with regard to cleaningefficiency and material composition (in particular the potential presence of toxic impurities orthe formation of reaction products with edible fats and oils).

• Potentially relevant impurities in the previous cargo should be taken into account, since theymay be toxicologically more important than the substance itself. As most products exist indifferent grades, a reasonable worst-case product within the specification provided wasassumed, the concentration of the impurity estimated from available sources and evaluated in

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the same way as a listed substance. Impurities are often specified for fine chemicals and highlypurified products. However, these are unlikely to be shipped in bulk. Those more commonlyencountered are likely to be of intermediate to low purity grade and no specific informationabout impurities is publicly available (sources and methods of synthesis are usuallyconfidential). Due to this lack of information, the source and most probable way (or ways) ofsynthesis of the substance are investigated to determine potentially relevant impurities, suchas unreacted starting substances or products of side reactions.

3. Assessment

3.1. Production and use of calcium lignosulfonate

Calcium lignosulfonate is obtained from sulfite pulping of wood (Toledo and Kuznesof, 2008). Fromthe three main components of wood, namely cellulose, hemicellulose and lignin, cellulose is isolated bydigesting the wood chips with acidic calcium bisulfite solution for 6–10 h at approximately 130°C.Bisulfite ions react with the native lignin, cleaving it to smaller sulfonated units. By this processlignosulfonate is formed, the water solubility of which enables its removal. Further reactions may causeelimination of water (Rydholm, 1965; Hoyt and Goheen, 1971). The hemicellulose is largely hydrolysedto monomeric sugars. The water-insoluble cellulose is separated by filtration. The brownish filtrate, thecrude lignosulfonate, consists of a complex mixture of breakdown products, but also includes naturalcomponents of wood, such as lipids, fatty acids, wax esters, sterols and their degradation products,resin acids and long-chain alcohols, as well as sulfite, inorganic salts, sugars and reaction products.

Technical grade products of lignosulfonates are steam-stripped, a process that removes the excessof sulfur dioxide (SO2) as well as volatile substances such as formaldehyde, furfural, hydroxymethylfurfural (HMF), acetic acid and formic acid. Fermentation followed by distillation of ethanol is a processthat may be used to remove the fermentable sugars. Subsequent water evaporation to solutions of50% lignosulfonate, the commercial products shipped, further reduces the volatile components.Precipitated salts are removed by filtration. Purification to obtain food- and feed-grade productsinvolves ultrafiltration through a semi-permeable membrane in order to further reduce the lowmolecular weight species (EFSA ANS Panel, 2010).

The largest use of lignosulfonates is as plasticiser in concrete to improve flow properties and slowsolidification. Lignosulfonates allow concrete to be made with less water (giving stronger concrete),while maintaining the flow properties. They are also applied during the production of cement, wherethey act as grinding aids. Similarly, lignosulfonates serve in the production of plasterboard to reducethe amount of water required to make the stucco flow and form the layer between two sheets ofpaper. The reduction in water content allows lower temperatures for drying, saving energy.

Calcium lignosulfonates are also used in petroleum drilling (blocking agent, improvement of mudfluidity), for asphalt emulsification, tanning leather, as a dispersant of chemicals and pesticides, as anadditive of slurry mixture of water and coal, and as an additive for feedstuff processing (deflocculant).

Further purified lignosulfonate products (calcium, sodium or magnesium salts) are also used as feedadditives (EFSA FEEDAP Panel, 2015).

3.2. Previous evaluations

Calcium lignosulfonate has been evaluated by the SCF in 1996 and considered acceptable asprevious cargo, being toxicologically inert and easily removable by tank cleaning. It was also evaluatedas an acceptable additive to animal feedstuff (SCF, 1997).

In 2008, calcium lignosulfonate 40–65 (a purified lignosulfonate product with an average molecularweight range of 40,000–65,000 Da) was evaluated by the Joint FAO/WHO Expert Committee on FoodAdditives (JECFA) as a food additive, intended to be used as carrier of encapsulated food ingredients(JECFA, 2009). JECFA established an ADI of 0–20 mg/kg bw based on a no-observed-effect level(NOEL) of 2,000 mg/kg bw per day from a 90-day dietary rat study and applying a safety factor of 100(JECFA, 2009).

In 2010, the EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS Panel)evaluated the use of calcium lignosulfonate 40–65 as a carrier for vitamins and carotenoids added tofoods for colouring and nutrient purposes (EFSA ANS Panel, 2010). It was specified that more than90% of this calcium lignosulfonate ranged between 1,000 and 250,000 Da, which was in agreementwith JECFA (2008). The ANS Panel concluded that the available data were insufficient to establish anADI and that long-term toxicity studies were needed to determine whether with time the histiocytosis

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in the mesenteric lymph nodes of the rats observed in the 90-day study may progress into a moreadverse state (EFSA ANS Panel, 2010).

In 2011, the ANS Panel considered that new information provided by the petitioner did not addressthe questions raised by the Panel in 2010 and that the requested chronic toxicity study of at least 12months was still needed (EFSA ANS Panel, 2011).

In 2011, the CONTAM Panel evaluated calcium lignosulfonate (unspecified, i.e. including lesspurified grades) as a acceptable previous cargo. Several data gaps (long-term toxicity, carcinogenicity,and limited data on reproductive toxicity) were noted. No evidence of genotoxicity or concernregarding allergenicity was reported in the limited data provided. The CONTAM Panel considered thatthe available information was sufficient to conclude that the exposure to the evaluated grade ofcalcium lignosulfonate (calcium lignosulfonate 40–65) would not give rise to toxicological concern whenused as a previous cargo. However, there were doubts that this evaluation would also cover the lesspurified grades mainly intended as previous cargoes, and no information on its potential reactivity withfats and oils was available. Therefore, the CONTAM Panel concluded that calcium lignosulfonate doesnot meet the criteria for acceptability as a previous cargo (EFSA CONTAM Panel, 2011).

In 2015, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) of EFSAdelivered an opinion on the safety of lignosulfonate for target animals, consumers, users and theenvironment, when used as a technological additive in feeds belonging to the functional group binders(EFSA FEEDAP Panel, 2015). It concluded that there was no concern for consumer safety from the usein feeds at up to 1% in complete feed. Since no margin of safety could be identified, this conclusionwas not extended to all animal species/categories. Lignosulfonate was neither considered as an irritantto the skin and eyes nor as a skin sensitiser. Exposure to dust by inhalation was considered a hazard.The FEEDAP Panel could not conclude on the safety of lignosulfonate for the environment.

In 2017, the CONTAM Panel re-evaluated calcium lignosulfonate as previous cargo, followingadditional information provided to EFSA. It still concluded that it does not meet the criteria as previouscargo due to data gaps, mainly regarding the composition and toxicity of the LMWF < 1,000 Da in thegrades of lignosulfonate intended as previous cargo.

3.3. Current evaluation

3.3.1. Additional information received

New data on genotoxicity were provided for a LMWF isolated from a technical product as intendedfor shipping. Later it became clear from the molecular mass distribution of the tested LMWF that thatthis did not contain a fraction of < 200 Da. The tests comprised a reverse mutation assay in bacteriaand an in vitro micronucleus assay in human peripheral lymphocytes. In addition, Borregaard providedan Ames test and an acute toxicity tests on some commercial lignosulfonate products.

Borregaard clarified that the calcium lignosulfonate products intended to be shipped as previouscargo are steam-stripped, but usually not further purified. Borregaard provided also data on themolecular mass distribution for the LMWF, the raw calcium lignosulfonate product from which theLMWF was isolated, a typical product intended for previous cargo, and the lignosulfonate 45–60purified by ultrafiltration, evaluated in the past as food and feed additive.

3.3.2. Composition of the low molecular weight fraction used for toxicity studies

According to Borregaard, the LMWF tested in the genotoxicity studies was isolated from arepresentative product of the grades intended to be shipped as previous cargo. It was obtained byultrafiltration through a membrane with molecular weight cut-off of 1,000 g/mol and freeze-drying.

This LMWF was obtained and characterised as follows:

‘A technical grade calcium lignosulfonate product was subjected to an ultrafiltration procedure usinga membrane with a MW cut-off of 1,000 Da followed by freeze-drying of the permeate. From a totalweight of 617 g lignosulfonate dry matter content a low molecular weight fraction of 35 g was isolatedas a brown powder. This corresponds to an isolated LMWF of 6% relative to the source material.

The purity of this LMWF was calculated using an indirect determination based on inorganic materialand total sugars according to the following formula, with all values expressed on a dry matter basis):

% lignosulfonate = 100% – (% total sugars) – (% inorganic material)

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For total sugars, a High-Performance Anion-Exchange Chromatography method with PulsedAmperometric Detection (HPAEC-PAD) was used, determining arabinose, galactose, rhamnose, glucose,xylose, fructose and mannose and expressing the total sugar content as the sum of these 7 sugars, Forinorganic material, the sample was combusted at 900°C and the ash determined gravimetrically.

Using this approach the LMWF was found to contain 56.8% calcium lignosulfonate with theremainder, by definition, being sugars and inorganic material. The doses (concentrations) applied inthe toxicity tests were increased by this factor of 100/56.8 (i.e. 91.76) so that the doses(concentrations) applied refer to the calcium lignosulfonate and not the total dry matter content’.

The LMWF was isolated from a raw (i.e. non-purified) lignosulfonate from a spruce raw material. Itwas not clear whether the product corresponds to the typical product intended for previous cargo, asin Table 1 the two products differ somewhat. However, it was stated that the products intended to beshipped are all manufactured from spruce as raw material and will have similar composition as theones presented and used for toxicity testing.

Specifically, the products intended to be shipped as previous cargo have similar molecular weightdistributions, with typical weight average molecular weight in the range of 30–40 kDa. However, it wasnot clearly specified in which respect these products differ (e.g. method of production, composition, etc.).

Borregaard provided mass distribution data for the isolated LMWF used in the genotoxicity testing, theraw lignosulfonate product from which the LMWF was isolated, a typical product intended for previouscargo and the lignosulfonate 45–60 (Table 1). The raw material used to isolate the tested LMWF wasintended to represent the least purified grades of product and had not been ultrafiltrated (the procedureused to reduce the amount of low molecular components for some lignosulfonate products).

The molecular weight distribution of the LMWF and the commercial grades of lignosulfonate wasdetermined using a size exclusion chromatography method with ultraviolet detection at 280 nm (SEC-UV).Chromatograms with absorbance vs. elution time were obtained and the relationship between retentiontime and MWwas calculated from a calibration curve/equation made using lignosulfonate standards of MW6,000 and 33,500 Da. The provided interpretation of the chromatograms is shown in Table 1.

According to this analysis, the LMWF tested contained 25.3% material below 1,000 Da, whichcorresponds to an enrichment by a factor of 2.8 and 3.1 compared to the raw lignosulfonate and thetypical product, respectively, used as reference products. However, the molecular mass distributionwithin the fraction < 1,000 Da deviated from that of the reference products: it mainly containedcompounds between 500 and 2,000 Da. The proportion of the fraction < 500 Da was only similar tothe two reference products and again with a distribution shifted towards the higher masses: thefraction < 200 Da was largely absent in the LMWF (0.1%).

The Panel concluded that the fraction tested for genotoxicity has not been shown to be sufficientlyrepresentative for the LMWF in the products intended to be shipped as previous cargo due to anapparent severe loss of constituents of lowest molecular mass. It was also noted that this analysis onlyrefers to components detected in UV at 280 nm, which mainly records phenol-type compounds, suchas lignin degradation products. According to Borregaard, 23 and 20% of the fraction tested consistedof inorganic salts and sugars, respectively, derived from cleaved hemicellulose. However, as thismaterial was not detected by UV at 280 nm and the mass distribution of these undetected constituentsis probably different from that of the material detected, it is unknown how this influenced thepercentages reported above. The Panel assumed that also the undetected compounds below 200 Dawere largely missing.

Table 1: Molecular mass distribution obtained by SEC-UV (280 nm) of the LMWF used forgenotoxicity testing (LMWF), the raw lignosulfonate from which the LMWF was isolated, alignosulfonate typical for the products intended as previous cargo and a lignosulfonatepurified by ultrafiltration (LS 40-65)

MW g/mol LMWF Raw material Typical product LS 40-65

< 6,000 100.0 45.3 42.9 30.6

< 2,000 83.9 20.6 18.7 7.8< 1,000 25.3 9.1 8.1 2.8

< 500 3.2 3.1 2.8 1.2

< 200 0.1 0.7 0.4 0.2

MW: molecular weight; LMWF: low molecular weight fraction; SEC-UV: size exclusion chromatography method with ultravioletdetection.

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Table 1 also provides data on the molecular mass distribution of a lignosulfonate product purified byultrafiltration (LS 40-65 evaluated in the past). According to the SEC-UV (280 nm) analysis, this productstill included 2.8% material < 1,000 Da, which corresponds to a reduction by a factor of around 3compared to the raw lignosulfonate and the typical product. Furthermore, the data suggests that themolecular mass distribution within the fraction < 1,000 Da was similar to the raw lignosulfonate and thetypical product, i.e. no selectivity in the removal by ultrafiltration is observed. However, not allpurification steps were disclosed that may have altered the composition of the LS 40-65.

3.3.3. Toxicity testing

The data available on the toxicity of calcium lignosulfonate are limited to the grade 40–65 productthat was more refined than the technical grades expected to be shipped in large amounts.

No new relevant data have been identified in the literature since the publication of the previousopinions of the CONTAM Panel (EFSA CONTAM Panel, 2011, 2017) regarding absorption, distribution,metabolism and elimination (ADME), acute toxicity and subacute, subchronic, chronic toxicity, in vitroand in vivo genotoxicity, carcinogenicity, developmental and reproductive toxicity, immunotoxicity andallergenicity.

In reaction to the previous evaluation of calcium lignosulfonate as a previous cargo (EFSA CONTAMPanel, 2017), Borregaard provided additional data on in vitro genotoxicity of a LMWF as described inSection 3.3.2. The genotoxicity testing strategy was in accordance with the recommendations of theEFSA Scientific Committee (2011).

In vitro genotoxicity with the LMWF

• Reverse mutation assay in bacteria

The LMWF has been tested, at doses of 0, 31.6, 100, 316, 1,000, 3,160 and 5,000 lg per plate ina set of five Salmonella Typhimurium strains TA98, TA100, TA102, TA1535 and TA 1537 in presence orabsence exogenous metabolic activation with S9 mix (rat liver induced by Aroclor 1254). No increase inrevertant colony numbers as compared with control numbers was observed in any of the five teststrains with and without metabolic activation. The positive controls showed a significant increase in thenumber of revertant colonies of the respective test strain (for details see Appendix A).

• Micronucleus assay

The capacity of LMWF to induce structural and/or numerical chromosome aberrations was tested incultured freshly isolated human peripheral lymphocytes (unspecified number of donors), in presence orabsence of metabolic activation, at concentrations of 0, 250, 500, 1,000 and 2,000 lg/mL. No increasein the frequency of micronucleated PCE was observed in presence of absence of S9 mix (rat liverinduced by Aroclor 1254). No signs of cytotoxicity were noted up to the highest concentration testedwith or without metabolic activation. The positive controls MMC and CP gave the expected positiveresults (for details see Appendix A).

In conclusion, the low molecular weight fraction (< 1,000 g/mol) of a standard commercial gradecalcium lignosulfonate was negative in two in vitro genotoxicity tests including a reverse mutationassay in bacteria and a micronucleus test in mammalian cells.

Other toxicity tests with technical grades of lignosulfonate

Borregaard also provided some additional toxicity data on certain of its products summarised inAppendix B.

Out of the five additional tests provided, only the Ames test might be of some relevance for thegenotoxicity testing of calcium lignosulfonate. However, in the absence (a) of further information ofthe product tested and a demonstration of its similarity with the products intended to be shipped and(b) of adequate set of in vitro genotoxicity assays (EFSA Scientific Committee, 2011, 2017, 2019), therelevance of this toxicity data for the evaluation of calcium lignosulfonate is questionable.

4. Conclusion

The data provided did not show the presence of the fraction < 200 Da in the material tested forgenotoxicity. The Panel concluded that the information provided was insufficient to assess theacceptability of lignosulfonate as previous cargo.

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5. Recommendations

To meet the criteria for acceptance as previous cargo, a better analysis of the toxicologically mostrelevant LMWF < 1,000 Da, including components < 200 Da, is needed. In particular, mass calibrationshould be performed with lower molecular mass standards, and a more comprehensive detectionshould be used.

Genotoxicity tests in accordance with EFSA guidance (Ames test and in vitro micronucleus test)(EFSA Scientific Committee, 2011, 2017, 2019) should be performed using the LMWF includingcomponents < 200 Da.

The tested material should be representative of the LMWF in products intended to be shipped asprevious cargo for edible fat and oils.

Documentation as provided to EFSA (if appropriate)

1) LPT Report No 36604 and No 36605. January 2019. Submitted by Borregaard.2) Letter to EFSA. August 2019. Submitted by Borregaard.3) Additional information. September 2019. Submitted by Borregaard.4) Reply to EFSA’s request. September 2016. Submitted by Borregaard.

ReferencesCCFO (Codex Committee on Fats and Oils), 2009. Report of the 21st CCFO meeting. Available online: http://

www.codexalimentarius.net/download/report/718/al32_17e.pdf [Accessed February 2016]EFSA (European Food Safety Authority), 2009. Scientific Opinion of the Panel on Contaminants in the Food Chain

on a request from the European Commission on the review of the criteria for acceptable previous cargoes foredible fats and oils. EFSA Journal 2009;7(5):1110, 21 pp. https://doi.org/10.2903/j.efsa.2009.1110

EFSA ANS Panel (EFSA Panel on Food Additives and Nutrient Sources Added to Food), 2010. Scientific opinion onthe use of calcium lignosulfonate (40-65) as a carrier for vitamins and carotenoids. EFSA Journal 2010;8(3):1525, 24 pp. https://doi.org/10.2903/j.efsa.2010.1525

EFSA ANS Panel (EFSA Panel on Food Additives and Nutrient Sources added to Food), 2011. Statement on thesafety of calcium lignosulfonate (40-65) as a food additive. EFSA Journal 2011;9(7):2319, 10 pp. https://doi.org/10.2903/j.efsa.2011.2319

EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), 2011. Scientific Opinion on the evaluationof the substances currently on the list in the Annex to Commission Directive 96/3/EC as acceptable previouscargoes for edible fats and oils–Part I of III. EFSA Journal 2011;9(12):2482, 61 pp. https://doi.org/10.2903/j.efsa.2011.2482

EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), 2012a. Scientific Opinion on the evaluationof the substances currently on the list in the annex to Commission Directive 96/3/EC as acceptable previouscargoes for edible fats and oils - Part II of III. EFSA Journal 2012a;10(5):2703, 151 pp. https://doi.org/10.2903/j.efsa.2012.2703

EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), 2012b. Scientific Opinion on the evaluationof the substances currently on the list in the annex to Commission Directive 96/3/EC as acceptable previouscargoes for edible fats and oils–Part III of III. EFSA Journal 2012b;10(12):2984, 82 pp. https://doi.org/10.2903/j.efsa.2012.2984

EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), Knutsen HK, Alexander J, Barreg�ard L,Bignami M, Br€uschweiler B, Ceccatelli S, Cottrill B, Dinovi M, Edler L, Hogstrand C, Hoogenboom LR, Nebbia C,Oswald I, Petersen A, Rose M, Roudot A-C, Schwerdtle T, Vollmer G, Wallace H, Grasl-Kraupp B, Grob K,Penninks A, Binaglia M, Rold�an Torres R and Vleminckx C, 2017. Scientific opinion on the evaluation ofsubstances as acceptable previous cargoes for edible fats and oils. EFSA Journal 2017;15(1):4656, 36 pp.https://doi.org/10.2903/j.efsa.2017.4656

EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), 2015. ScientificOpinion on the safety and efficacy of lignosulfonate as a feed additive for all animal species. EFSA Journal2015;13(7):4160, 17 pp. https://doi.org/10.2903/j.efsa.2015.4160

EFSA Scientific Committee, 2011. Scientific Opinion on genotoxicity testing strategies applicable to food and feedsafety assessment. EFSA Journal 2011;9(9):2379, 69 pp. https://doi.org/10.2903/j.efsa.2011.2379

EFSA Scientific Committee, Hardy A, Benford D, Halldorsson T, Jeger M, Knutsen HK, More S, Naegeli H, NotebornH, Ockleford C, Ricci A, Rychen G, Silano V, Solecki R, Turck D, Younes M, Aquilina G, Crebelli R, G€urtler R,Hirsch-Ernst KI, Mosesso P, Nielsen E, van Benthem J, Carf�ı M, Georgiadis N, Maurici D, Parra Morte J andSchlatter J, 2017. Scientific Opinion on the clarification of some aspects related to genotoxicity assessment.EFSA Journal 2017;15(12):5113, 25 pp. https://doi.org/10.2903/j.efsa.2017.5113

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EFSA Scientific Committee, More S, Bampidis V, Benford D, Boesten J, Bragard C,Halldorsson T, Hernandez-JerezA, Hougaard-Bennekou S, Koutsoumanis K, Naegeli H, Nielsen SS, Schrenk D, Silano V, Turck D, Younes M,Aquilina G, Crebelli R, G€urtler R, Hirsch-Ernst KI, Mosesso P, Nielsen E, Solecki R, Carfi M, Martino C, Maurici D,Parra Morte J and Schlatter J, 2019. Statement on the genotoxicity assessment of chemical mixtures. EFSAJournal 2019;17(1):5519, 11 pp. https://doi.org/10.2903/j.efsa.2019.5519

FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization), 2011. Joint FAO/WHOFood Standards Programme Codex Alimentarius Commission. 34th Session International Conference Centre.Geneva, Switzerland, 4-9 July 2011. Report. REP11/CAC. Available online: http://www.who.int/foodsafety/codex/34thCAC.pdf

Hoyt CH and Goheen DW, 1971. Polymeric Products. Chapter 20. In: Sarkanen KV, Ludvig CH(eds.). Lignins;Occurrence, formation, structure and reactions. Wiley-Interscience, New York, US, pp. 846–857.

JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2008. New specifications prepared at the 69thJECFA (2008), published in FAO JECFA Monographs 5. Available online: http://www.fao.org/3/a-i0345e.pdf

JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2009. Safety evaluation of certain food additives.WHO Food Additives Series 60, 69th meeting of JECFA. World Health Organization, Geneva. Available online:http://apps.who.int/iris/bitstream/10665/44063/1/9789241660600_eng.pdf

OECD TN, 1997. 471: bacterial reverse mutation test. OECD Guidelines for the Testing of Chemicals, Section, 4.OECD, 2016. Test N. 487 (2016): In vitro mammalian cell micronucleus test. OECD guidelines for the testing of

chemicals, section 4.OECD TG401 Acute Oral Toxicity OECD Guidelines for the Testing of Chemicals, Section, 4.OECD TG404 Acute dermal irritation/corrosion. OECD Guidelines for the Testing of Chemicals, Section, 4.OECD TG405 Acute eye irritation/corrosion OECD Guidelines for the Testing of Chemicals, Section, 4.OECD TG429 Skin sensitisation: local lymph node assay. OECD Guidelines for the Testing of Chemicals, Section, 4.Rydholm SA, 1965. Pulping processes. Interscience Publishers, New York, US, 1269 pp.SCF (Scientific Committee on Food), 1997. Opinion on the potential risk to human health arising from the transport

in ships’ tanks of oils and fats from substances proposed as acceptable previous cargoes (expressed on 20September 1996). Annex VII to Document III/5693/96. DG III, European Commission, Brussels.

SCF (Scientific Committee on Food), 2003. Updated opinion of the Scientific Committee on Food on the potentialrisk to human health arising from the transport in ships’ tanks of oils and fats from substances proposed asacceptable previous cargoes (expressed on 4 April 2003). Health and Consumer Protection Directorate-General,European Commission, Brussels.

Toledo MCF and Kuznesof PM, 2008. Calcium Lignosulphonate (40-65) Chemical and Technical Assessment, draftCTA provided by DSM Nutritional Products, Basel Switzerland for the 69th JECFA meeting. Available online:http://www.fao.org/fileadmin/templates/agns/pdf/jecfa/cta/69/Calcium_Lignosulphonate__40_65.pdf

Abbreviations

ADI acceptable daily intakeADME absorption, distribution, metabolism and eliminationANS EFSA Panel on Food Additives and Nutrient Sources Added to FoodCAC Codex Alimentarius CommitteeCCFO Codex Committee for Fats and OilsCONTAM EFSA Panel on Contaminants in FoodECHA European Chemicals AgencyFEEDAP EFSA Panel on Additives and Products or Substances used in Animal FeedGLP Good Laboratory PracticeHPAEC-PAD high-performance anion-exchange chromatography method with pulsed amperometric

detectionJECFA The Joint FAO/WHO Expert Committee on Food AdditivesLD50 lethal dose, 50%LMWF low-molecular weight fractionNOEL no-observed-effect levelOECD Organisation for Economic Co-operation and DevelopmentSCF Scientific Committee on FoodSEC-UV size exclusion chromatography method with ultraviolet detectionTDI total daily intakeWHO World Health Organization

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Appendix A – In vitro genotoxicity with the LMWF

Table A.1: In vitro genotoxicity tests on calcium lignosulfonate low molecular weight fraction(< 1,000 g/mol) (LMWF)

Type of testExperimentaltest system

Test substanceExposureconditions

Results Comments

Reversemutationassay

SalmonellaTyphimuriumTA98, TA100,TA1535, TA1537and TA102

Calciumlignosulfonate

LMWF (< 1,000g/mole)

Batch No. DP3678

56.8% (calciumlignosulfonatefraction)

Solvent =negative control:aqua adinjectabilia

Positive controls:positivesubstances asrecommended inTG 471

With andwithout S9 mix3 plates/conc.

Preliminary test:plateincorporationassay in TA100

10 conc.: 0.316–5,000 lg/plate

Main test:1st exp.: plateincorporationassay

2nd exp.: pre-incubation test6 conc.: 31.6–5,000 lg/plate

No signs ofcytotoxicity up to5,000 lg/plateNegative

Bacteria colonieswere counted usingthe Biosys Biocount5000 system

GLP-compliantassay in accordanceto OECD 471(1997)

Micronucleustest

Humanlymphocytes

Calciumlignosulfonate

LMWF (< 1,000g/mol)

Batch No. DP3678

56.8% (calciumlignosulfonatefraction)

Solvent =negative control:aqua adinjectabilia

Positive controls:–S9: MMC andcolchicine, +S9:CP

With andwithout S9 mix(rat liver inducedby Aroclor 1254

Preliminarycytotoxicity test:7 conc.: 3.16–2,000 lg/mL,expo: –S9: 4 hand 24 h, +S9:4 h

Main test:1st exp.: – S9: 4h expo.,harvesting 20 hlater, 250, 500and 2,000 lg/mL; + S9 (2% v/v): 4 h expo.,harvesting 20 hlater, 250, 500and 2,000 lg/mL

2nd exp.: –S9:24 h expo.,harvesting atend of treatment

Duplicatecultures

No signs ofcytotoxicity up to2,000 lg/mL

Negative

No relevantchanges on pH orosmolality

Cytokinesis blocktechnique applied(Cyto B)

GLP-compliantassay in accordancewith OECD 487(2016)

OECD: Organisation for Economic Co-operation and Development; GLP: Good laboratory practice.

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Appendix B – Other toxicity tests with technical grades of lignosulfonate

Table B.2: Ames test

Type oftest

Experimentaltest system

Test substance Exposure conditions Result Comments

Reversemutationassay

SalmonellaTyphimurium

TA98, TA100,TA1535, TA1537

Calciumlignosulfonate

Positive controls:B(a)P and Naazide

With and without S9mix (rat liver inducedby Aroclor 1254)1st test: 0, 0.01, 0.1, 1and 10 mg/plate2nd. test: 0, 0.1, 0.5, 1and 5 mg/platePlate incorporationassay

Negative Not according to GLPBefore OECD 471 wasdeveloped

OECD: Organisation for Economic Co-operation and Development; GLP: Good laboratory practice.

Table B.1: Acute toxicity

Test Substance tested Result Comments

Acute oral toxicity

Rat (Wistar)

5 M + 5 F/group

Sodium lignosulfonate LD50 > 2,000 mg/kg bw According to OECD TG401

GLP

Skin irritation

Rabbit (4)

Calcium lignosulfonate Not irritant According to OECD TG404

GLP

Eye irritation

Rabbit (4)

Calcium lignosulfonate Not irritant According to OECD TG405

GLP

Skin sensitisation

Local lymph nodeassay in mouse

Calcium lignosulfonate

(composition provided)

Not sensitiser According to OECD TG429

GLP

LD50: lethal dose, 50%; bw: body weight; OECD: Organisation for Economic Co-operation and Development; GLP: GoodLaboratory Practice.

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Annex A – Review of the criteria for acceptable previous cargoes for ediblefats and oils (EFSA, 2009)

In 2009, EFSA received a request from the European Commission to review the criteria foracceptable previous cargoes for edible fats and oils set by the SCF (Table A.1). The CONTAMPanel assessed the appropriateness of the four criteria of the Codex Committee for Fats and Oils(CCFO) (Table A.2) by comparing them with those set by the SCF in 1996 (EFSA, 2009).

‘The CONTAM Panel concluded that the criteria for evaluation of acceptable previous cargoes foredible fats and oils as proposed by the CCFO are not in conflict with any of the five criteria developedby SCF. SCF criteria 1 to 4 are either explicitly or implicitly covered by the CCFO criteria. SCF criterion5, dealing with the availability of analytical methods, is not explicitly addressed in the CCFO criteria.The CONTAM Panel considers that SCF criterion 5 is still important. The CCFO criteria also cover foodallergens and compounds that may react with oil and fats. The CONTAM Panel considers theseadditions relevant. In addition, the CONTAM Panel made the following remarks:

• The CCFO criteria specifically apply to the immediate previous cargo. The CCFO criterion 1,which addresses, among other issues, documentation procedures, does not specify for howmany previous cargoes records should be kept. This might be particularly important in theevent that earlier previous cargoes consist of substances for which an acceptable daily intake(ADI) (or tolerable daily intake, TDI) has not been established. The CONTAM Panel was of theopinion that records of the three previous cargoes should be kept, in accordance with theCodex Recommended International Code of Practice for the Storage and Transport of EdibleOils and Fats in Bulk.

• The CCFO criteria specifically apply to the immediate previous cargo. The CCFO criterion 1,which addresses, among other issues, documentation procedures, does not specify for how

Table A.1: Criteria for the inclusion of substances in the list of acceptable previous cargoesaccording to the SCF (1997, 2003)

SCF Criteria(a)

1. No toxicological concerns, particularly with regard to their genotoxic and carcinogenic potential, for which athreshold is difficult to establish

2. Efficacy of procedures used to clean ships’ tanks between cargoes3. Dilution factor in relation to the potential amount of residue of the previous cargo and any impurity which

the previous cargo might have contained and the quantity of oil or fat transported

4. Subsequent application of refining processes and solubility relevant to the occurrence of possiblecontaminating residues

5. Availability of analytical methods to verify the presence of trace amounts of residues or the absence ofcontamination of oils and fats

(a): The SCF criteria have no numbering in the original reference.

Table A.2: Criteria proposed for immediate previous cargoes by the Codex Committee for Fats andOils (CCFO) during their 21st meeting (CCFO, 2009) and adopted by the CodexAlimentarius Committee (CAC) (FAO/WHO, 2011)

CCFO Criteria (adopted at Step 5)

1. The substance is transported/stored in an appropriately designed system; with adequate cleaning routines,including the verification of the efficacy of cleaning between cargoes, followed by effective inspection andrecording procedures

2. Residues of the substance in the subsequent cargo of fat or oil should not result in adverse human healtheffects. The ADI (or TDI) of the substance should be greater than or equal to 0.1 mg/kg bw/day.Substances for which there is no numerical ADI (or TDI) should be evaluated on a case by case basis

3. The substance should not be or contain a known food allergen, unless the identified food allergen can beadequately removed by subsequent processing of the fat or oil for its intended use

4. Most substances do not react with edible fats and oils under normal shipping and storage conditions.However, if the substance does react with edible fats and oils, any known reaction products must complywith criteria 2 and 3

ADI: acceptable daily intake; TDI: total daily intake; bw: body weight.

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many previous cargoes records should be kept. This might be particularly important in theevent that earlier previous cargoes consist of substances for which an acceptable daily intake(ADI) (or tolerable daily intake, TDI) has not been established. The CONTAM Panel was of theopinion that records of the three previous cargoes should be kept, in accordance with theCodex Recommended International Code of Practice for the Storage and Transport of EdibleOils and Fats in Bulk.

• With respect to CCFO criterion 2, the EFSA Panel on Contaminants in the Food Chain (CONTAMPanel) agreed with the proposed threshold of an ADI (or TDI) of ≥ 0.1 mg/kg body weight(bw). For substances for which there is no numerical ADI (or TDI) a case by case evaluation isneeded. The Panel also considered the situation of second and third previous cargoes andconcluded that for non-genotoxic substances their transport as second and third previouscargoes is not of concern, taking into account their very limited carry over. However, theCONTAM Panel noted that genotoxic substances would not be acceptable as previous cargoes.Also, in relation to CCFO criterion 2, the CONTAM Panel noted that as consequence of theabove some substances will turn out to be unacceptable as previous cargoes. This couldinclude substances with ADI (or TDI) < 0.1 mg/kg bw or substances with genotoxic activity.The Panel was of the opinion that the exclusion of such substances as previous cargoes isappropriate.

• CCFO criterion 3 is sufficient to cover “known food allergens”. However, the CONTAMPanel considered that the scope of the CCFO criterion is too narrow, and should apply to allknown allergens, not just to known food allergens, given the fact that the same cargo may besold for cosmetic use.

• The CONTAM Panel endorsed CCFO criterion 4 without further remarks’.

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